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MALS验证的高生物活性重组三聚体蛋白

背景
具有正确折叠和构象的重组蛋白才能有效保证抗体药物研发的前期免疫、抗体筛选和鉴定、候选抗体药物体外功能验证、抗体药物质量控制以及临床样本分析等过程的顺利进行。蛋白质只有在正确的聚体结构下才能有效激活下游通路,发挥其生理功能。比如,在调控免疫应答过程中发挥重要的作用的肿瘤坏死因子(TNF)超家族,该系列蛋白在天然状态下为三聚体结构,只有当三聚体形式TNF-α与TNFR1的胞外区结合时才能引起TNFR1的三聚化,进而有效的激发下游通路引起细胞凋亡。蛋白质的聚集状态是决定其功能和活性的重要性质之一,我们不仅要在重组蛋白设计、表达和生产过程中严谨以待,还需要建立准确可靠的分子量测定分析方法来表征蛋白的聚集状态。

图1.SEC-MALS检测原理图
a.光散射示意图 b.分析物分子量计算公式
常见的蛋白分子量测定技术有相对定量法(SEC-HPLC,高效分子筛液相色谱)和绝对定量法(SEC-MALS,多角度激光散射),两种技术都可以测定溶液中蛋白的分子量,但由于原理上的不同,导致检测结果准确度存在显著差异(表1)。SEC-HPLC需要借助标准蛋白进行分子量内插测定,实际应用中常常受到多种因素的干扰,无法准确得到蛋白质真实分子量和聚集状态。MALS技术是基于蛋白的分子量与光散射的强度直接相关来测定蛋白绝对分子量的技术,无需使用标准蛋白,不依赖于洗脱体积。通过SEC分离后的各组分进入多角光散射检测器,激光照射到分析物时会发生光的散射,MALS通过多个角度同时测定散射光的强度(图1.a),公式b1可以看出散射光强度正比于摩尔质量、浓度、折光指数增量的平方,且通过用公式b2可以直接计算出分析物的绝对分子量。
为满足市场需求,ACROBiosystems已经开发了25种由HEK293表达的经过MALS验证的TNF超家族分子蛋白,ACRO提供的TNF系列蛋白适用于大分子抗体药物的免疫、SPR、细胞活性检测等实验,更有AvitagTM生物素标记的蛋白可以助力于您的药物开发。
表1. SEC-HPLC和SEC-MALS两种检测方法的比较
检测方法 原理和特点 结果
SEC-HPLC 柱校正法
  • 假设待测蛋白与标准蛋白的构象,形状相似
  • 假设与柱填料无任何非特异的相互作用
  • 假设每次洗脱时间可重现无漂移
通过比较待测蛋白和标准蛋白的洗脱保留时间内插计算相对分子量
SEC-MALS 光散射法
  • 散射光强度正比于摩尔质量、浓度、折光指数增量的平方
  • 散射光角度依赖性仅与分子尺寸大小相关
通过激光散射强度直接测定绝对分子量,分子量测定不依赖于洗脱体积或保留时间
产品特点
SEC-MALS验证蛋白的真实分子量和聚体状态
OXL-H52Q8
Human OX40 Ligand /TNFSF4 Protein, His Tag, active trimer

Fig.1 The purity of Human OX40 Ligand, His Tag, active trimer (Cat. No. OXL-H52Q8) was more than 95% and around 60-80 kDa verified by SEC-MALS.

Protocol

CDL-H5266
Human CD27 Ligand / CD70 Protein, Fc Tag, active trimer

Fig.2 The purity of Human CD27 Ligand, Fc Tag, active trimer (MALS verified, Cat. No. CDL-H5266) was more than 85% and around 160-190 kDa verified by SEC-MALS.

Protocol

蛋白的生物活性由细胞实验、SPR、ELISA多种技术的验证
CDL-H5269
Human CD40 Ligand / TNFSF5 Protein, Fc Tag, active trimer

Fig.3 FACS analysis shows that Human CD40 Ligand, Fc Tag, active trimer (MALS verified) (Cat. No. CDL-H5269) can bind to 293T-CD40 cells surface CD40. The concentration of Human CD40 Ligand is 0.3 μg/mL.

Protocol

TNA-H4211
ActiveMax® Human TNF-alpha Protein, Tag Free, active trimer

Fig.4 ActiveMax® Human TNF-alpha, Tag Free, low endotoxin (active trimer) (MALS verified) (Cat. No. TNA-H4211) induces cytotoxicity effect on the WEH1-13VAR cells in the presence of the metabolic inhibitor actinomycin D. The EC50 for this effect is 0.007-0.014 ng/mL.

Protocol

41L-H52D4
Human 4-1BB Ligand / TNFSF9 Protein, His,Flag Tag, active trimer

Fig.5 Human 4-1BB Ligand (71-254), His,Flag Tag, active trimer (MALS verified) (Cat. No. 41L-H52D4) induce IL-8 secretion in HT1080 human CD137 cell line. The EC50 for this effect is 0.20-0.44 μg/mL.

Protocol

LIT-H5242
Human LIGHT /TNFSF14 Protein, His Tag, active trimer

Fig.6 Human LIGHT, His Tag, active trimer (MALS verified) (Cat. No. LIT-H5242) induced cytotoxicity in HT-29 cells. The ED50 for this effect is 1.11-3.87 ng/mL.

Protocol

RAL-H5240
Human TNFSF11 /RANKL /CD254 Protein, His Tag, active trimer

Fig.7 Captured Human RANK, Mouse IgG2a Fc Tag, low endotoxin (Cat. No. RAK-H5251) on CM5 chip via Anti-Mouse antibodies surface can bind Human TNFSF11, His Tag, active trimer (Cat. No. RAL-H5240) with an affinity constant of 0.309 nM as determined in a SPR assay (Biacore T200).

Protocol

CDL-H5266
Human CD27 Ligand / CD70 Protein, Fc Tag, active trimer

Fig.8 Immobilized Human CD27, His Tag (Cat. No. CD7-H522b) at 2 μg/mL (100 μL/well) can bind Human CD27 Ligand, Fc Tag, active trimer (MALS verified) (Cat. No. CDL-H5266) with a linear range of 0.2-8 ng/mL.

Protocol

产品列表
MALS验证的TNF超家族产品列表
Molecule Cat. No. Species Product Description
4-1BB Ligand 41L-H5265 Human Human 4-1BB Ligand / TNFSF9 (71-254) Protein, Fc Tag (active trimer) (MALS verified)Hot
41L-H52D4 Human Human 4-1BB Ligand / TNFSF9 (71-254) Protein, His,Flag Tag (active trimer) (MALS verified)
APRIL APL-H52D1 Human Human BAFF / TNFSF13B / CD257 Protein, Fc Tag, active trimer (MALS verified)Hot
BAFF BAF-H5261 Human Human BAFF / TNFSF13B / CD257 Protein, Fc Tag, active trimer (MALS verified)
BAF-H52D4 Human Human BAFF / TNFSF13B / CD257 Protein, His,Flag Tag, active trimer (MALS verified)
BAF-H82F3 Human Biotinylated Human BAFF / TNFSF13B / CD257 Protein, Avitag™,Fc Tag (active trimer) (MALS verified)
BAF-H82Q2 Human Biotinylated Human BAFF / TNFSF13B / CD257 Protein, His,Avitag™ (active trimer) (MALS verified)
CD27 Ligand CDL-H5266 Human Human CD27 Ligand / CD70 Protein, Fc Tag (active trimer) (MALS verified)Hot
CDL-H52Da Human Human CD27 Ligand / CD70 Protein, His,Flag Tag (active trimer) (MALS verified)
CDL-H82D7 Human Biotinylated Human CD27 Ligand / CD70 Protein, His,Avitag™,Flag Tag (active trimer) (MALS verified)
CDL-C5244 Cynomolgus Cynomolgus CD27 Ligand / CD70 Protein, His Tag (active trimer) (MALS verified)
CDL-M5245 Mouse Mouse CD27 Ligand / CD70 Protein, His Tag (active trimer) (MALS verified)
CD40 Ligand CDL-H5269 Human Human CD40 Ligand / TNFSF5 Protein, Fc Tag (active trimer) (MALS verified)Hot
CDL-H52Db Human Human CD40 Ligand / TNFSF5 Protein, His,Flag Tag (active trimer) (MALS verified)
CDL-M5248 Mouse Mouse CD40 Ligand / TNFSF5 Protein, His Tag (active trimer) (MALS verified)
Fas Ligand FAL-H5241 Human Human Fas Ligand / TNFSF6 Protein, His Tag (active trimer) (MALS verified)
LIGHT LIT-H5242 Human Human LIGHT / TNFSF14 Protein, His Tag (active trimer) (MALS verified)
LIT-H5269 Human Human LIGHT / TNFSF14 Protein, Fc Tag (active trimer) (MALS verified)Hot
OX40 Ligand OXL-H52Q8 Human Human OX40 Ligand / TNFSF4 Protein, His Tag (active trimer) (MALS verified)
TNF-alpha TNA-H4211 Human ActiveMax® Human TNF-alpha Protein, Tag Free, low endotoxin (active trimer) (MALS verified)
TNA-H5228 Human Human TNF-alpha Protein, His Tag (active trimer) (MALS verified)
TNA-H8211 Human Biotinylated Human TNF-alpha Protein, epitope tag free, ultra sensitivity (primary amine labeling) (active trimer) (MALS verified)
TNA-H82E3 Human Biotinylated Human TNF-alpha Protein, His,Avitag™ (active trimer) (MALS verified)
TNA-M82E9 Mouse Biotinylated Mouse TNF-alpha Protein, His,Avitag™ (active trimer) (MALS verified)
TNFSF11 RAL-H5240 Human Human TNFSF11 / RANKL / CD254 Protein, His Tag (active trimer) (MALS verified)
RAL-H82Q5 Human Biotinylated Human TNFSF11 / RANKL / CD254 Protein, His,Avitag™, active trimer (MALS verified)
MALS验证的其他天然三聚体蛋白
Molecule Cat. No. Species Product Description
S protein SPN-C52H8 2019-nCoV 2019-nCoV (COVID-19) Full Length S protein (R683A, R685A) , His Tag, active trimer (MALS verified)

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